Currently available models for toxicity screening are not always accurate predictors of toxicity in humans. Animal models are commonly used, but they are costly, time-consuming, and ethically challenging, they vary between species, and they do not accurately predict toxicity in humans. In vitro toxicity tests have been explored for years as cheaper alternatives or as initial screens before in vivo testing, but there are still issues regarding accuracy, primarily because they are cultured on two-dimensional (2D) surfaces, while native tissues exist in three-dimensional environments (3D). As a result, while ethical and cost motivations drive toxicity screening towards in vitro models, the limitations of current in viro assays in mimicking native tissue have prevented their widespread acceptance and use. This proposal puts forward a 3D model that is rapid, quantitative, and representative for high-throughput toxicity testing. Recently, research has gravitated towards in vitro three-dimensional (3D) cell culture systems, which are more representative in native tissue environment and responses than 2D systems, and still less costly and controversial than in vivo tests. The advantages of 3D include: (1) dynamic spatial gradients of soluble factor concentrations; (2) a wider array of cell-cell and cell-matrix interactions that regulate cell function and behavior differently; and (3) the ability to support multiple cell types with spatial organization to mimic native environments. As a result, 3D cell culture models for toxicity testing could represent native tissue environments and predict in vivo toxicity better than 2D in vitro models. However, currently available 3D cell culture models are not ideal given that these models are expensive, involve extensive fabrication, and are time-consuming to analyze. For example, in one comparable model, 3D spheroids took 7-10 days to assay cytotoxicity. The long experimentation time of these 3D in vitro assays limits the number of compounds studied while increasing risks related to cell culture, like contamination. To that end, this proposal looks to design a 3D human cell-based in vitro assay that better represents the human tissue of interest, predicts in vivo toxicity, but does so within a shorter timeframe than other assays. We use magnetic nanoparticles, which are nontoxic, and can be used to render cells magnetic. These magnetized cells can then be manipulated with magnetic forces with fine spatial control, and without the need of any special equipment or media. In this proposal, we will use this technique to print cells into 3D rings, that we have found to close/contract over time, and at rates that vary with compound concentration. This allows for the easy and rapid printing of 3D cellular models for the purpose of toxicity screening. Additionally, we will use a mobile device-based imaging system to image whole plates, and in doing so, increasing efficiency and throughput of the assay at a significantly lower cost. In Phase I, we propose to validate our model with 3T3 mouse embryonic fibroblasts, which are commonly used for toxicity testing, according to NIEHS guidelines, before expanding into organ-specific toxicity models, specifically of the lung and liver, in Phase II. The resulting 3D toxicity assay from this proposal will use the advantages of 3D cell culture to better predict in vitro toxicity in a quick, cost-effective fashion. In addition this proposal will develop mobile- device based analytical tools for high-throughput analysis. Our hypothesis is that we can design a novel in vitro 3D assay using magnetic printing that applies the benefits of 3D cell culture towards toxicity screening that better predicts in vivo toxicity thn other in vitro assays. These assays would yield fast, quantitative, label-free metrics of cell migration under different conditions to study the basal cytotoxicity of certain compounds. This assay would also allow for high-throughput analysis to improve screening throughput and efficiency. In addition, post-assay experimentation, including fluorescent staining, can be performed on the 3D cultures to investigate particular mechanisms of action. In creating a magnetically printed 3D assay, we will integrate: Capability to rapidly print 3D cell cultures with relevant extracellular matrix; Real-time and label-free quantification of ring closure, which correlates with cell function; Ability to investigate the basal cytotoxicity of particular compound and their mechanisms of actions; Tools for high-throughput analysis that could significantly cut the time and cost of data collection. The end result of this project is an assay that is faster tha other assays, less costly than animal models and 3D cultures, and more predictive than 2D in vitro assays. This proposal has letters of support from researchers at University of Texas Health Science Center - Houston, University of Texas MD Anderson Cancer Center, Rice University, Genentech, and AstraZeneca. The aims of this Phase I SBIR proposal are: Aim I: Optimize the magnetic levitation and patterning of 3D cell cultures for the BiO Assay Aim II: Validate the BiO Assay for measuring cytotoxicity Aim III: Validate the mobile device-based image acquisition in the BiO Assay.